JP7601913B2 - Protein, hemolytic streptococcus vaccine, DNA, vector, transformant, method for producing protein, baculovirus, agrobacterium, latex particle, kit, and method for measuring anti-streptolysin O antibody - Google Patents

Description

The present invention relates to proteins, hemolytic streptococcal vaccines, DNA, vectors, transformants, methods for producing proteins, baculoviruses, agrobacteria, latex particles, kits, and methods for measuring anti-streptolysin O antibodies.

Streptolysin O (SLO) is a protein with hemolytic activity produced by Streptococcus pyogenes. When humans are infected with Streptococcus pyogenes, anti-SLO antibodies (ASO) in the blood increase. Using this, the presence or absence of infection with Streptococcus pyogenes is clinically examined by measuring the amount of ASO in the blood. SLO is used as a raw material for ASO measurement reagents.

SLO is a protein with a total length of 571 amino acid residues, with the peptide from the N-terminus to the 33rd amino acid residue being the signal peptide. SLO34-571, which does not contain the signal peptide, has been used as a raw material for ASO measurement reagents. In recent years, modified SLO with a deleted C-terminal region has been developed with the aim of providing an SLO that has no hemolytic activity and is highly thermostable (Patent Document 1).

International Publication No. 2017/204325

The location of the epitope of a neutralizing antibody against SLO in SLO has not yet been studied. The inventors considered that a protein containing an epitope of a neutralizing antibody against SLO can be used as a vaccine antigen capable of producing neutralizing antibodies, and can also be used as a test antigen capable of quantifying neutralizing antibody titers. In other words, the object of the present invention is to determine the location of the epitope of a neutralizing antibody against SLO, and to provide a protein (SLO antigen) containing such an epitope.

The inventors investigated the reactivity of neutralizing antibodies against SLO and found that it was 100% against SLO82-571 and approximately 70% against SLO34-460 and SLO82-460, making it clear that SLO82-460 is the major epitope of neutralizing antibodies against SLO. Based on these findings, the inventors have completed the present invention.

That is, the present invention relates to the following [1] to [21].
[1] Any protein selected from the following (a) to (g):
(a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; (b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (c) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4; (d) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, in which a portion of the amino acid residues at positions 1 to 48 have been deleted; (e) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, in which a portion of the amino acid residues at positions 380 to 490 have been deleted; (f) a protein consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence of any of the proteins (a) to (e), and which binds to ASO; (g) a protein having a tag attached to at least one of the C-terminus and N-terminus of any of the proteins (a) to (f).
[2] Any protein selected from the following (a') to (e'):
(a') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; (b') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (c') a protein consisting of an amino acid sequence in which a portion of the amino acid residues at positions 1 to 48 in the amino acid sequence set forth in SEQ ID NO: 2 have been deleted; (d') a protein consisting of an amino acid sequence that has 90% or more identity with the amino acid sequence of any of the proteins (a') to (c') and binds to ASO; (e') a protein having a tag attached to at least one of the C-terminus and N-terminus of any of the proteins (a') to (d').
[3] The protein according to [1] or [2], wherein the tag is a His tag.
[4] A diagnostic antigen for measuring a neutralizing antibody that neutralizes the hemolytic activity of streptolysin O, comprising the protein according to any one of [1] to [3] above.
[5] A hemolytic streptococcal vaccine comprising the protein according to any one of [1] to [3] above.
[6] A DNA encoding the protein according to any one of [1] to [3] above.
[7] A vector comprising the DNA described in [6] above.
[8] A transformant comprising the vector according to [7] above.
[9] The transformant according to [8] above, wherein the transformant is selected from bacteria, insect cells, insects, animal cells, animals, plant cells and plants.
[10] A method for producing the protein according to any one of [1] to [3] above, comprising a step of culturing, raising or cultivating the transformant according to [8] or [9] above.
[11] A baculovirus comprising the vector according to [7] above integrated into bacmid DNA.
[12] A method for producing the protein according to any one of [1] to [3] above, comprising a step of infecting a host insect cell or insect with the baculovirus according to [11] above.
[13] A method for producing the protein according to any one of [1] to [3] above, wherein the vector according to [7] above is a viral vector, the method comprising a step of infecting a host animal cell or animal with the vector.
[14] An Agrobacterium comprising the vector according to [7] above.
[15] A method for producing the protein according to any one of [1] to [3] above, comprising a step of infecting a host plant with the Agrobacterium according to [14] above.
[16] Latex particles having the protein according to any one of [1] to [3] above adsorbed thereon.
[17] A kit for measuring ASO, comprising the latex particles according to [16] above.
[18] The kit described in [17] above, wherein the ASO is a neutralizing antibody that neutralizes the hemolytic activity of SLO.
[19] A method for measuring ASO using the latex particles described in [16] above.
[20] A step of contacting the latex particles according to the above-mentioned [16] with a test sample that may contain ASO, and a step of measuring an agglutination reaction of the latex particles due to an antigen-antibody reaction between the antibody and the protein.
A method for measuring an anti-SLO antibody, comprising:
[21] The method according to [19] or [20] above, wherein the ASO is a neutralizing antibody that neutralizes the hemolytic activity of SLO.

According to the present invention, it is possible to provide a vaccine antigen capable of producing neutralizing antibodies against SLO, and a protein (SLO antigen) that can be used as a testing antigen capable of quantifying neutralizing antibody titer against SLO.

1 shows the results of SDS-PAGE of each SLO variant under non-reducing conditions, with the leftmost lane being molecular weight marker, lane 1 being SLO34-460, lane 2 being SLO82-460, and lane 3 being SLO82-571. 1 shows the results of an antibody absorption test using SLO34-460, in which (a) shows the results when ASO-positive serum before antibody absorption was added as a ligand, and (b) shows the results when the flow-through fraction after antibody absorption was added as a ligand. 1 shows the results of an antibody absorption test using SLO82-460, in which (a) shows the results when ASO-positive serum before antibody absorption was added as a ligand, and (b) shows the results when the flow-through fraction after antibody absorption was added as a ligand.

The following describes in detail an embodiment of the present invention.

One embodiment of the present invention is a protein selected from the following (a) to (g): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; (b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2; (c) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4; (d) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which a portion of the amino acid residues at positions 1 to 48 has been deleted; (e) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which a portion of the amino acid residues at positions 380 to 490 has been deleted; (f) a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of any of the proteins (a) to (e) and binding to ASO; or (g) a protein having a tag attached to at least one of the C-terminus and N-terminus of any of the proteins (a) to (f). Such a protein contains an epitope of a neutralizing antibody against SLO, and can therefore be a vaccine antigen capable of producing a neutralizing antibody.

SLO34-571, which is obtained by removing amino acid residues 1 to 33 (signal peptide) from full-length SLO, is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. SLO34-460, which is obtained by removing amino acid residues 1 to 33 and 461 to 571 from full-length SLO, is a "protein consisting of the amino acid sequence shown in SEQ ID NO: 2." SLO82-460, which is obtained by removing amino acid residues 1 to 81 and 461 to 571 from full-length SLO, is a "protein consisting of the amino acid sequence shown in SEQ ID NO: 3." SLO82-571, which is obtained by removing amino acid residues 1 to 81 from full-length SLO, is a "protein consisting of the amino acid sequence shown in SEQ ID NO: 4."

"A protein consisting of an amino acid sequence in which some of the amino acid residues at positions 1 to 48 in the amino acid sequence set forth in SEQ ID NO:2 have been deleted" corresponds to a protein in which some of the amino acid residues at positions 34 to 81 of full-length SLO have been deleted from SLO34-460. The positions and number of the deleted amino acid residues are not limited, and any one or more amino acid residues in the range of positions 1 to 48 may be deleted.

"A protein consisting of an amino acid sequence in which a portion of the amino acid residues at positions 380 to 490 in the amino acid sequence set forth in SEQ ID NO: 4 has been deleted" corresponds to a protein in which a portion of the amino acid residues at positions 461 to 571 of full-length SLO has been deleted from SLO82-571. The positions and number of the deleted amino acid residues are not limited, and any one or more amino acid residues in the range of positions 461 to 571 may be deleted.

The identity of the amino acid sequence in a "protein that has an amino acid sequence that is 90% or more identical to the amino acid sequence of any of proteins (a) to (e) and binds to ASO" may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be less than 100%. The method of determining whether a protein binds to an ASO is an immunological measurement method on a detection substrate on which a protein is immobilized, and includes an ELISA method, an immunochromatography method, an immunoagglutination method (latex agglutination (LA) method), a chemiluminescent immunoassay (CLIA) method, a pulse immunoassay method, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, a QCM (Quartz Crystal Microbalance) method, a BioLayer Interferometry (BioLayer Interferometry) method, and a ELISA method. Examples of the immunoassay include, but are not limited to, a turbidimetric immunoassay (TIA) method, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, a QCM (Quartz Crystal Microbalance) method, a BioLayer Interferometry (BLI) method, and a surface plasmon resonance method. Examples of the immunoassay include, but are not limited to, a turbidimetric immunoassay (TIA) method, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, a QCM (Quartz Crystal Microbalance) method, a BioLayer Interferometry (BLI) method, and a surface plasmon resonance method. Here, to determine whether or not a protein binds to ASO, a test statistic calculated from two metric values obtained when a protein is applied to a test group (i) that does not contain ASO and a test group (ii) that may contain ASO is used to test the difference between the population means of two populations. The test statistic may be the difference obtained by subtracting the measured value of (i) from the measured value of (ii), or the value obtained by dividing the measured value of (ii) by the measured value of (i), but the test statistic to be adopted is not limited to this. Therefore, when the null hypothesis H ₀ : μ _i = μ _ii , the alternative hypothesis H ₁ : μ _i ≠ μ _ii , and the significance level α = 0.05 are set using the test statistic, if the test statistic t ₀ is in the rejection region, H ₀ is rejected, and it is determined that the protein and ASO are bound to each other.

The tag in "a protein having a tag added to at least one of the C-terminus and N-terminus of any of the proteins (a) to (f)" is a peptide or protein added for the purpose of isolating, purifying, immobilizing, detecting, solubilizing, visualizing, etc. of the protein. For example, solubilization tags include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), and Nus tags, and detection tags include S tags, HSV tags, FLAG tags, His tags, Strep tags, Strep-II tags, Myc tags, HA tags, V5 tags, E tags, T7 tags, VSV-G tags, Glu-Glu tags, and Avi tags. The His tags, Strep tags, and Strep-II tags can also be used as purification tags. Proteins to which these tags have been added can be expressed as fusion proteins. Proteins to which these tags have been added can be used as they are as vaccine antigens capable of producing neutralizing antibodies. Furthermore, proteins to which these tags have been added can be treated with a digestive enzyme to cleave the tags, thereby obtaining any of the proteins (a) to (f). Among these tags, the His tag is preferred. The His tag can be used for purifying proteins by utilizing its property of specifically binding to metal ions such as nickel. The number of histidine residues contained in the His tag may be, for example, 4 to 10, 5 to 7, or 6.

The protein is preferably any of the proteins selected from the following (a') to (e'): (a') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, (b') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, (c') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which a portion of the amino acid residues at positions 1 to 48 have been deleted, (d') a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of any of the proteins (a') to (c') and binding to ASO, and (e') a protein having a tag attached to at least one of the C-terminus and N-terminus of any of the proteins (a') to (d'). Such a protein contains SLO82-460, which is the main epitope of neutralizing antibodies against SLO, and is therefore particularly useful as a vaccine antigen capable of producing neutralizing antibodies.

One embodiment of the present invention is a diagnostic antigen for measuring a neutralizing antibody that neutralizes the hemolytic action of SLO, which is composed of the above protein. As described below in the kit for measuring ASO and the method for measuring ASO, the above protein can be used as a diagnostic antigen for measuring the above neutralizing antibody. The above diagnostic antigen can be used to qualitatively evaluate the presence or absence of the above neutralizing antibody in a test sample, or to quantitatively evaluate the concentration of the above neutralizing antibody in a test sample. If the neutralizing antibody ability of a subject can be measured, it may be possible to predict the severity of the subject and the risk of reinfection.

One embodiment of the present invention is a hemolytic streptococcus vaccine comprising the above protein.

The vaccine may be in the form of, for example, liquid, powder (lyophilized powder, dry powder), capsule, tablet, or frozen.

The vaccine may contain a medicamentously acceptable carrier. The carrier may be any carrier commonly used in vaccine production without limitation. Specifically, the carrier may include saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer, and combinations thereof. The vaccine may further contain an emulsifier, a preservative (e.g., thimerosal), an isotonicity agent, a pH adjuster, an inactivating agent (e.g., formalin), etc., as appropriate.

The route of administration of the vaccine may be, for example, transdermal, sublingual, ophthalmic, intradermal, intramuscular, oral, enteral, nasal, intravenous, subcutaneous, intraperitoneal, or by inhalation from the mouth to the lungs.

The vaccine may be administered, for example, by syringe, transdermal patch, microneedle, implantable sustained release device, syringe connected to microneedle, needleless device, or spray.

To further enhance the immunogenicity of the vaccine, the vaccine may contain an adjuvant together with the protein. Examples of adjuvants include aluminum adjuvants or oil-in-water emulsion adjuvants containing squalene (AS03, MF59, etc.), Toll-like receptor ligands such as CpG and 3-O-deacylated-4'-monophosphoryl lipid A (MPL), saponin-based adjuvants, polymer-based adjuvants such as poly-γ-glutamic acid, and polysaccharides such as chitosan and inulin.

Mammals that are the target of the vaccine of this embodiment include, for example, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, humans, etc. The vaccine of this embodiment may be used for humans, children under the age of 5, and elderly people over 65.

One embodiment of the present invention is a DNA that encodes the above protein. By using such a DNA, the above protein can be produced.

One embodiment of the present invention is a vector containing the above DNA.

Examples of vectors include plasmids, viruses, cosmids, lambda phages, and artificial chromosomes. Examples of viruses include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and baculoviruses. The vector is preferably an expression vector containing a promoter sequence, etc.

When the expression host is a eukaryote, it is preferable that the vector has a promoter sequence, a ribosome binding sequence, a translation initiation site, a polyadenylation tail, a Kozak sequence, etc.

When the expression host is a prokaryote such as a bacterium, it is preferable that the vector is capable of autonomous replication in the prokaryote and has a promoter sequence, a ribosome binding sequence, a translation initiation site, etc.

In addition to these, vectors may also contain, for example, replication origins, genetic markers, antibiotic resistance, epitopes, targeting sequences, and reporter genes.

One embodiment of the present invention is a transformant containing the above vector.

The transformant may be, for example, a prokaryote such as a bacterium, a yeast, a filamentous fungus, an insect, an animal, an algae, a plant, or a cell thereof. The transformant is preferably selected from a bacterium, an insect cell, an insect, an animal cell, an animal, a plant cell, and a plant.

Examples of bacteria include Escherichia coli, Brevibacillus bacteria, Corynebacterium bacteria, and Rhodococcus bacteria.

Examples of yeasts include those belonging to the genera Saccharomyces, Pichia, Candida, and Torulopsis.

Examples of filamentous fungi include those belonging to the genera Aspergillus, Penicillium, and Trichoderma.

Examples of insects include insects of the Noctuidae and Bombycidae families of the Lepidoptera order.

Examples of animals include mammals, and examples of animal cells include cells derived from human cervical cancer, human fetal kidney, hamster ovary, and African green monkey kidney.

Examples of algae include algae belonging to the genera Chlamydomonas and Synechococcus.

Examples of plants include plants belonging to the Solanaceae family (e.g., tobacco, eggplant, tomato, bell pepper, chili pepper), Rosaceae family (e.g., rose, strawberry), Brassicaceae family (e.g., Arabidopsis thaliana, rapeseed, Chinese cabbage, cabbage, radish, rapeseed), Asteraceae family (e.g., chrysanthemum, garland chrysanthemum, lettuce), Chenopodiaceae family (e.g., spinach, sugar beet), Poaceae family (e.g., wheat, rice, barley, corn) and Legume family (e.g., soybean, adzuki bean, kidney bean, broad bean). For example, the plant body may be a plant of the Solanaceae or Brassicaceae family, a plant of the Nicotiana genus or an Arabidopsis genus, such as Nicotiana benthamiana, Nicotiana tabacum, or Arabidopsis thaliana.

Methods for transforming an expression host include, for example, electroporation, a method using calcium ions, the spheroplast method, the protoplast method, the lithium acetate method, the calcium phosphate method, the lipofection method, and the competent cell method. Methods for expressing a heterologous protein using a plant as an expression host include, for example, the agroinfiltration method, the plant virus vector method, the magnICON (registered trademark) system, and the particle gun method.

The vector may be directly transformed into an expression host, or the vector may be transformed into any cell and the cell may be used to transform into an expression host. Examples of such cells include bacteria, yeast, and animal cells. For example, the protein can be expressed in an expression host or an expression host cell by infecting the expression host or the expression host cell with a transformed bacterium.

When the transformant is a plant, examples of the bacteria into which the vector is introduced include bacteria of the species Agrobacterium, Rhizobium, Sinorhizobium, Mesorhizobium, Phyllobacterium, Ochrobactrum, and Bradyrhizobium, and Agrobacterium is preferred. That is, one embodiment of the present invention is an Agrobacterium containing the above-mentioned vector.

The case where the plant is a transformant will be described in more detail below. The method of expressing the above protein using a plant as an expression host can be carried out by a known method. Such methods include, for example, the agroinfiltration method, the plant virus vector method, the magnICON (registered trademark) system, and the particle gun method.

When using the agroinfiltration method, Agrobacterium can be transformed with the above vector and the Agrobacterium can be infected into a plant body to obtain a plant body that expresses the above protein.

When using the plant virus vector method, a plant expressing the above protein can be obtained as follows. First, RNA is obtained from the cDNA of the plant virus genome into which DNA encoding the above protein has been inserted. Then, this RNA is inoculated as a vector into the plant to infect it. Examples of such virus vectors include tobacco mosaic virus (TMV) vectors, plum pox virus (PPV) vectors, potato virus X (PVX) vectors, alfalfa mosaic virus (AIMV) vectors, cucumber mosaic virus (CMV) vectors, cowpea mosaic virus (CPMV) vectors, and zucchini yellow mosaic virus (ZYMV) vectors.

When using the magnICON® system, a plant expressing the above protein can be obtained as follows. First, the cDNA of the TMV or PVX genome into which the DNA encoding the above protein has been inserted is introduced into a T-DNA vector. Next, the plant is infected with Agrobacterium transformed with the obtained T-DNA vector.

When the transformant is an insect or insect cell, a baculovirus having the above vector incorporated into bacmid DNA can be used. That is, one embodiment of the present invention is a baculovirus having the above vector incorporated into bacmid DNA. Bacmid DNA is a shuttle vector that can be replicated in an insect or insect cell and E. coli, and is DNA in which a bacterial artificial chromosome (BAC) sequence is introduced into the genomic DNA of a baculovirus that grows in an insect so that it can replicate autonomously in E. coli. The above vector is incorporated into the bacmid DNA of the baculovirus of this embodiment. If the bacmid DNA is purified from E. coli and transfected into an insect cell, it can grow in the insect cell as a baculovirus.

One embodiment of the present invention is a method for producing the protein, comprising the step of culturing, raising or cultivating the transformant. By cultivating, raising or cultivating the transformant, the protein can be expressed within the transformant.

The manufacturing method of this embodiment may further include a step of extracting the expressed protein. Protein extraction can be performed by known methods, such as physical disruption (homogenizer, bead mill, ultrasonic, French press, etc.), osmotic shock, freeze-thaw, addition of surfactant, and enzyme digestion.

The manufacturing method of this embodiment may further include a step of isolating, purifying and/or concentrating the extracted protein. The isolation, purification and/or concentration of the protein can be performed, for example, by affinity, ion exchange, gel filtration and hydrophobic interaction chromatography.

One embodiment of the present invention is a method for producing the above protein, comprising the step of infecting a host plant with the above Agrobacterium.

The production method of this embodiment may further include a step of cultivating the infected host plant. The production method of this embodiment may further include a step of extracting the expressed protein. The method of protein extraction is as described above. The production method of this embodiment may further include a step of isolating, purifying and/or concentrating the extracted protein. The method of protein isolation, purification and/or concentration is as described above.

One embodiment of the present invention is a method for producing the above protein, comprising the step of infecting a host insect cell or insect with the above baculovirus.

The production method of this embodiment may further include a step of culturing or rearing the infected host insect cell or insect. The production method of this embodiment may further include a step of extracting the expressed protein. The method of protein extraction is as described above. The production method of this embodiment may further include a step of isolating, purifying and/or concentrating the extracted protein. The method of protein isolation, purification and/or concentration is as described above.

One embodiment of the present invention is a method for producing the protein, comprising the step of infecting a host animal cell or animal with the vector, wherein the vector is a viral vector. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and baculoviruses.

The production method of this embodiment may further include a step of culturing or raising the infected host animal cell or animal. The production method of this embodiment may further include a step of extracting the expressed protein. The method of protein extraction is as described above. The production method of this embodiment may further include a step of isolating, purifying and/or concentrating the extracted protein. The method of protein isolation, purification and/or concentration is as described above.

One embodiment of the present invention is a latex particle having the above-mentioned protein adsorbed thereon.

Examples of materials for latex particles include polystyrene and divinylbenzene. The average particle size of latex particles can be 0.1 μm to 5 μm. The particle size of latex particles can be measured by dynamic light scattering. In this specification, "average particle size" refers to the particle size (median diameter) when the integrated value from the smallest particle size reaches 50% of the total in the volume-based particle size distribution curve obtained by dynamic light scattering.

The above proteins can be adsorbed onto latex particles using common physical adsorption methods.

One embodiment of the present invention is a kit for measuring ASO, comprising the above-mentioned latex particles.

The kit for measuring ASO can be used to qualitatively evaluate the presence or absence of ASO in a test sample, or to quantitatively evaluate the concentration of ASO in a test sample.

The kit for measuring ASO may further include a standard sample of ASO and a buffer solution for diluting the test sample.

It is preferable that the ASO to be measured is a neutralizing antibody that neutralizes the hemolytic action of SLO. Since the above protein contains an epitope of a neutralizing antibody against SLO, it is also possible to measure the neutralizing antibody titer.

One embodiment of the present invention is a method for measuring ASO using the above-mentioned latex particles.

The ASO measurement method can be performed to qualitatively evaluate the presence or absence of ASO in a test sample, or to quantitatively evaluate the concentration of ASO in a test sample.

In one embodiment, the measurement method includes a step of contacting the latex particles with a test sample that may contain ASO, and a step of measuring the agglutination reaction of the latex particles due to an antigen-antibody reaction between the antibody and the protein.

By mixing the suspension containing the latex particles with the test sample, the latex particles can be brought into contact with the test sample that may contain ASO. When the two are brought into contact, the latex particles aggregate due to the interaction between the ASO contained in the test sample and the protein, and the absorbance of the suspension changes. The amount of change in absorbance (endpoint method) or the rate of change (rate method) is measured. For the measurement, a turbidimetric method or a colorimetric method is preferably used. For example, the agglutination reaction of the latex particles is measured by irradiating light in the visible to near-infrared range, usually 300 nm to 1000 nm, preferably 500 nm to 900 nm, from the outside of the cell and detecting the change in absorbance or the change in the intensity of the scattered light.

The time for carrying out the agglutination reaction may be 1 to 30 minutes, preferably 1 to 10 minutes, but is not limited to these. The temperature for carrying out the agglutination reaction may be 35°C to 40°C, or may be 36 to 38°C, but is not limited to these.

A number of standard samples containing the ASO to be measured at various known concentrations are prepared, and the amount or rate of change in absorbance is measured for each of them using the above method. A calibration curve is drawn by plotting the concentration of ASO in the standard samples on the horizontal axis and the measured amount or rate of change in absorbance on the vertical axis. The amount or rate of change in absorbance of an unknown test sample is also measured using the same method, and the measurement results are applied to the above calibration curve, allowing the ASO in the test sample to be quantitatively evaluated. In addition, a threshold value for the amount or rate of change in absorbance is set in advance, and it can be qualitatively evaluated that ASO is present in the test sample if the threshold value is exceeded.

The test sample is not particularly limited as long as it is capable of containing the target antibody, but examples include body fluids such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, swabs, etc., or dilutions thereof, with blood, serum, plasma, urine, stool, cerebrospinal fluid, or dilutions thereof being preferred.

It is preferable that the ASO to be measured is a neutralizing antibody that neutralizes the hemolytic activity of SLO. Since the above protein contains an epitope of a neutralizing antibody against SLO, it is also possible to measure the neutralizing antibody titer.

Test Example 1. Preparation of SLO variants SLO variants lacking some of the amino acid residues of SLO and having the amino acid sequences shown in SEQ ID NOs: 2 to 4 were prepared by the following method.
SEQ ID NO: 2: SLO34-460
SEQ ID NO: 3: SLO82-460
SEQ ID NO: 4: SLO82-571

1.1. Expression and purification of SLO variants in plants SLO variants were produced using the plant transient expression system described in Japanese Patent Publication No. 5015012. Genes of SLO variants encoding SEQ ID NOs: 2 to 4 were cloned into a tobacco mosaic virus (TMV) vector (Icon Genetics). After introducing these vectors into Agrobacterium for transformation and cultivation, the mixture of culture solutions was infiltrated into Nicotiana benthamiana leaves and harvested in about one week. The harvested leaves (infected leaves) were frozen and then ground in a mortar. 30 ml of extraction buffer was added to 10 g of ground infected leaves and left on ice for 30 minutes. Then, the mixture was centrifuged at 15,000 x g and 4°C for 15 minutes, and the supernatant was collected. The collected supernatant was further centrifuged at 15,000 x g and 4°C for 10 minutes, and the supernatant was collected. The supernatant was applied to a Ni affinity column (His GraviTrap, Cytiva) under the following conditions, and the eluted fraction was collected. The collected eluted fraction was subjected to buffer exchange using a desalting column (Econo-Pac 10DG Desalting Columns, Bio-Rad Laboratories), and concentrated using an ultrafiltration column (Vivaspin Turbo 15, 10,000 MWCO PES, Sartorius).

Column volume (CV) = 1 mL
Steps: equilibration/10 CV, load/30 CV, wash/15 CV, elution/3 CV x 3
Extraction buffer: 20 mM phosphate buffer pH 7.4, 500 mM sodium chloride, 20 mM imidazole Column equilibration buffer: same as extraction buffer Elution buffer: 20 mM phosphate buffer pH 7.4, 500 mM sodium chloride, 500 mM imidazole The purification yield of each protein when each SLO variant was purified is shown in Table 1. As shown in Table 1, good expression was observed for all SLO variants. In addition, the yields of SLO34-460 and SLO82-460, which are SLO variants in which domain 4 of SLO (consisting of amino acid residues 461-571) has been removed, were 2.5 times or more higher than the yield of SLO82-571.

The results of SDS-PAGE of each SLO variant under non-reducing conditions are shown in Figure 1. As shown in Figure 1, each of the targeted SLO variants was detected as a major band, confirming that each SLO variant was purified to a high degree of purity.

Test Example 2. Antibody Absorption Test 2.1. Absorption of antibodies to immobilized SLO variants Each SLO variant was immobilized on an antigen immobilization column (Pierce NHS-Activated Agarose Spin Columns, Thermo Scientific), and an ASO-positive serum control (Immunocucela I-(H) "Seiken" RD, Denka) or an ASO-positive serum sample was added and reacted (antibody absorption) for 16 hours at 4°C. The flow-through fraction after the reaction was collected, and the buffer of the flow-through fraction was replaced with PBS using a desalting column (PD MiniTrap G-25, Cytiva).

2.2. Confirmation of antibody absorption to immobilized SLO variants Using the surface plasmon resonance (SPR) method, it was confirmed whether ASO was present in the pass-through fraction after antibody absorption. The SPR method was performed using a Biacore (Biacore 8K, Cytiva) equipped with a sensor chip (Series S Sensor Chip Protein A, Cytiva) on which Protein A was immobilized. When the ASO-positive serum before antibody absorption or the pass-through fraction of the SLO variant immobilized column after antibody absorption was added as a ligand and the antibody was bound to Protein A on the sensor chip, a response of about 3000 RU was obtained as the capture level for both. Furthermore, each SLO variant used for immobilization was added thereto at various concentrations as an analyte. The results when the SLO variants immobilized on the column and the analyte were SLO34-460 are shown in FIG. 2, and the results when the analyte was SLO82-460 are shown in FIG.

When ASO-positive serum before antibody absorption was added as a ligand, a concentration-dependent binding signal to the analyte was observed (Figures 2(a) and 3(a)). On the other hand, when the flow-through fraction of the SLO variant immobilized column after antibody absorption was added as a ligand, no binding signal was observed even when a relatively high concentration of 900 nM SLO variant was added (Figures 2(b) and 3(b)).

Here, the area of the immobilized portion on the sensor chip is 1.2 mm ² (Reference 1), and 1000 RU ≈ 1 ng/mm ² (p. 13 of Reference 2). Therefore, a response with a capture level of about 3000 RU means that the total amount of IgG captured on the sensor chip is 3.6 ng. If the molecular weight of IgG is 150 kDa, 3.6 ng of IgG is about 0.024 pmol. Furthermore, the unit of an analyte concentration of 900 nM is converted to 0.9 pmol/mm ^3. Since the volume of the reaction site of the sensor chip is 0.06 mm ³ (Reference 1), the amount of analyte present in the reaction site volume is 0.054 pmol. Therefore, when the concentration of the analyte, i.e., the SLO variant, is 900 nM, the SLO variant is present at the reaction site of the sensor chip in an amount more than twice as much as the antibody (ASO) against the SLO variant, which is sufficient to detect a binding signal. However, the fact that no binding was observed when the flow-through fraction of the SLO variant-immobilized column was added as a ligand indicates that the ASO was not present in the flow-through fraction, and there is no stoichiometric contradiction.

The step in the sensorgram when the analyte is added is due to the so-called solvent effect caused by the difference in refractive index between the analyte solvent (PBS) and the buffer (0.01 M HEPES, pH 7.4, 0.15 M sodium chloride, 3 mM EDTA, 0.05% Tween 20) (Reference 2, p. 64), and is a self-evident fact in the field of testing.

From the above results, it was confirmed that no antibodies (ASOs) that bind to the immobilized SLO mutant were present in the flow-through fraction of the SLO mutant immobilized column.

Test Example 3. Measurement of Neutralizing Antibody Titer 3.1. Preincubation of SLO variants and ASO SLO34-571 dissolved in PBS solution was added with DTT to 10 mM DTT, and incubated at room temperature (25°C) for 5 minutes to activate the SLO variant (Reference 3), and DTT was removed using an ultrafiltration column. Next, 80 μl of a solution prepared with PBS solution to give a concentration of 0.6 μg/ml of the activated SLO variant, and 80 μl of a solution of the ASO positive serum control or the flow-through fraction after antibody absorption, which were serially diluted with PBS solution, were added to a 96-well U-bottom plate and preincubated at 25°C for 1 hour.

3.2. Hemolysis test Rabbit defibrinated blood (Kohjin Bio Co., Ltd., catalog number 12065305) was washed with PBS to prepare 3% rabbit defibrinated blood. 80 μl of 3% rabbit defibrinated blood was added to the plate in which SLO34-571 and ASO were pre-incubated, and incubated at 37°C for 45 minutes. The plate was then centrifuged at 1000 xg for 5 minutes, and 200 μl of the supernatant was transferred to a flat-bottom plate for absorbance measurement, and the absorbance at 541 nm, which indicates the degree of hemolysis, was measured (References 4 and 5). The neutralizing ability (%) at each dilution step was calculated from the degree of hemolysis using the following formula.
Neutralizing ability (%)=100−{(sample−non-hemolytic control)/(hemolytic control−non-hemolytic control)}×100
Sample: absorbance ( _OD541 ) of each sample
Non-hemolytic control: SLO variant and ASO positive serum control, or absorbance (OD ₅₄₁ ) when PBS solution was added instead of the flow-through fraction after antibody absorption
Hemolysis control: absorbance (OD ₅₄₁ ) when PBS solution was added instead of ASO positive serum control or the flow-through fraction after antibody absorption

The dilution ratio (IC ₅₀ ) showing 50% neutralizing activity was calculated from the neutralizing activity (%) at each dilution step, and this was taken as the neutralizing antibody titer. Table 2 shows the neutralizing antibody titers before and after antibody absorption using the ASO positive serum control.

As is clear from the results shown in Table 2, the neutralizing activity of the flow-through fraction when SLO34-460 or SLO82-460 was immobilized was reduced by about 70% compared to the ASO-positive serum control before antibody absorption. Therefore, it can be said that the reactivity of neutralizing antibodies against each SLO variant is as shown in Table 3.

Similar experiments were performed using clinical samples (serum) known to be ASO positive instead of the ASO positive serum control, and similar results were obtained. These are shown in Tables 4 and 5.

Similar experiments were also carried out on other ASO-positive clinical specimens. As is clear from the results shown in Tables 6 and 7, SLO34-460 and SLO82-460 were shown to capture approximately 70% of neutralizing antibodies in ASO-positive clinical specimens with different neutralizing antibody titers (IC ₅₀ ).
The above results show that, among neutralizing antibodies against SLO, the abundance ratio of neutralizing antibodies having SLO34-460 and SLO82-460 as epitopes was constant at about 70% regardless of the specimen.
It is apparent from these results that the remaining 30% of the neutralizing antibodies are antibodies whose epitope is domain 4 (consisting of amino acid residues 461-571). Therefore, in Tables 6 and 7, the neutralizing activity (%) of the flow-through fraction upon immobilization of SLO82-571 and the reactivity of the neutralizing antibodies to SLO82-571 are not shown.

These results indicate that SLO34-460 and SLO82-460 are capable of capturing approximately 70% of the majority of neutralizing antibodies regardless of the specimen, and that the remaining 30% of neutralizing antibodies are antibodies that have domain 4 as an epitope. Therefore, it was demonstrated that SLO34-460 and SLO82-460 are not only useful for diagnosing hemolytic streptococcus infections, but are also the major epitopes of neutralizing antibodies that exhibit clinically important protective ability against hemolysis caused by hemolytic streptococcus infection. Therefore, it is believed that these antigens are also useful as vaccine antigens against hemolytic streptococcus.

References Reference 1: Morio Arai, "Analysis of biomolecular interactions using a biosensor (BIACORE) using surface plasmon resonance - Application to research in the field of thrombosis and hemostasis -", Journal of the Japanese Society on Thrombosis and Hemostasis, Vol. 8 (No. 5), pp. 397-405, 1997 Reference 2: Setsuko Hashimoto and Kaori Morimoto (eds.), "Interaction analysis using Biacore: Experimental method", Maruzen Publishing, 2012 Reference 3: BHAKDI, SUCHARIT, et al., "Isolation and identification of two hemolytic forms of streptolysin-O.", Infection and immunity, 1984, 46.2: 394-400.
Reference 4: DALE, James B., et al., “Antibodies against a synthetic peptide of SagA neutralize the cytolytic activity of streptolysin S from group A streptococci.”, Infection and immunity, 2002, 70.4: 2166-2170.
Reference 5: HUGO, FERDINAND, et al., “Use of a monoclonal antibody to determine the mode of transmembrane pore formation by streptolysin O.”, Infection and immunity, 1986, 54.3: 641-645.

Claims (20)

Any protein selected from the following (a) to (c):
(a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3; (b) a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of the protein (a) and which binds to an anti-streptolysin O antibody; (c) a protein having a tag attached to at least one of the C-terminus and N-terminus of any of the proteins (a) to (b). The protein of claim 1, wherein the tag is a His tag. A diagnostic antigen for measuring a neutralizing antibody that neutralizes the hemolytic activity of streptolysin O, comprising the protein according to claim 1 or 2. A hemolytic streptococcus vaccine comprising the protein according to claim 1 or 2. DNA encoding the protein according to claim 1 or 2. A vector comprising the DNA of claim 5. A transformant comprising the vector according to claim 6. The transformant according to claim 7 , wherein the transformant is selected from bacteria, insect cells, insects, animal cells, non-human animals, plant cells and plants. A method for producing the protein according to claim 1 or 2, comprising the steps of:
A method comprising a step of culturing, raising or cultivating the transformant according to claim 7 or 8. A baculovirus in which the vector according to claim 6 has been incorporated into bacmid DNA. A method for producing the protein according to claim 1 or 2, comprising the steps of:
A method comprising the step of infecting a host insect cell or insect with the baculovirus of claim 10. A method for producing the protein according to claim 1 or 2, comprising the steps of:
7. A method according to claim 6, wherein the vector is a viral vector, the method comprising a step of infecting a host animal cell or a non-human animal with the vector. An Agrobacterium comprising the vector according to claim 6. A method for producing the protein according to claim 1 or 2, comprising the steps of:
A method comprising a step of infecting a host plant with the Agrobacterium of claim 13. Latex particles to which the protein according to claim 1 or 2 is adsorbed. A kit for measuring anti-streptolysin O antibodies, comprising the latex particles according to claim 15. The kit according to claim 16, wherein the anti-streptolysin O antibody is a neutralizing antibody that neutralizes the hemolytic activity of streptolysin O. A method for measuring anti-streptolysin O antibodies using the latex particles according to claim 15. A step of contacting the latex particles according to claim 15 with a test sample that may contain an anti-streptolysin O antibody, and a step of measuring an agglutination reaction of the latex particles due to an antigen-antibody reaction between the antibody and the protein.
A method for measuring an anti-streptolysin O antibody, comprising: The measurement method according to claim 18 or 19, wherein the anti-streptolysin O antibody is a neutralizing antibody that neutralizes the hemolytic action of streptolysin O.